P436: CUTTING-EDGE TRANSCRIPTOMICS TO IDENTIFY LEUKEMIA-INTRINSIC AND -EXTRINSIC CORRELATES OF IMMUNE ESCAPE AND POST-TRANSPLANTATION RELAPSE
Authors: Pier Edoardo Rovatti, Marco Punta, Matteo Maria Naldini, Matteo Barcella, Laura Zito, Matteo Giovanni Carrabba, Massimo Bernardi, Jacopo Peccatori, Ivan Merelli, Bernhard Gentner, Fabio Ciceri, Luca Vago, Cristina Toffalori
Journal: HemaSphere (2023)

Background: Despite Allogeneic Hematopoietic Cell Transplantation (allo-HCT) being the most successful therapeutic option for many patients suffering from Acute Myeloid Leukemia (AML), between 40-60% of allo-HCT recipients experience disease reoccurence. Previous work demonstrated that AML relapses are often the result of an immune evasion process, where leukemic blasts, either by reducing antigen presentation or enforcing inhibitory checkpoint pathways, escape donor T cell recognition. Nonetheless, a sizable portion of disease recurrences are still unaccounted for. Many other immune cell types weave a canvas of interactions with leukemic clones in the bone marrow niche, where AML originates, and this complex tumor microenvironment (TME) has already been demonstrated to play a role in anti-tumor immunity. Aims: Here, we used single-cell RNA-sequencing (scRNA-seq) to map in detail the changes that occur in the bone marrow of patients who experience AML relapse after allo-HCT. Methods: As of today, we used the 10x Genomics Single Cell Immune Profiling solution to profile by scRNA-seq: 165,059 cells from 19 AML patients with different mechanisms of leukemia relapse, including downregulation of HLA class II (n=5), upregulation of inhibitory ligands (n=4), and genomic loss of incompatible HLAs (“HLA loss”) (n=10); 28,066 cells from 4 healthy donors and 52,698 cells from 4 post-transplant patients in complete remission. Results: First, by quantification of allele-specific HLA expression (scHLAcount) and using a selected list of genes involved in HLA class II and inhibitory ligand surface expression, we were able to recapitulate the distinctive features of each relapse modality. Based on the observation that AML relapses can arise from distinct cell populations, ranging from more stem/progenitor phenotypes to immunophenotypically committed, we used three gene signatures that represent successive stages of normal hematopoietic development (HSC/Prog, GMP, and differentiated myeloid) to calculate a gene module score that could estimate the degree of maturation of each relapse modality. Interestingly, we observed that HLA loss relapses have a more immature expression profile compared to the other relapse modalities (Fig. 1), which also resulted in a higher LSC17 score, suggesting that the hematopoietic cell of origin impacts on the mechanism of post-transplantation relapse. Focusing on the TME, we exploited souporcell to identify host- and donor derived cells for each sample in our cohort, allowing us to not only separate healthy progenitor and leukemic cells, but also highlight the presence of a previously unknown host-derived CD14+CD16- mature monocyte cluster exhibiting an anti-inflammatory gene signature, which might play a facilitating role in leukemia immune evasion. Finally, we leveraged on TCR sequencing to dissect the T cell immune repertoire complexity in the different relapse settings. CD8+ T cells, in particular effector memory (TEM) and terminally differentiated TEM showed higher clonality in AML BM. We then used the Shannon index, a robust metric of clonal heterogeneity, to quantify the TCR repertoire diversity for each patient in a given experimental group. Interestingly, AML relapses characterized by MHC-II downregulation displayed a lower Shannon index, and therefore a more clonal architecture, as compared to other relapse mechanisms. Summary/Conclusion: Findings from this study will improve our understanding of how leukemic cells exploit their TME to escape immune surveillance in the different patients and identify new vulnerabilities to be exploited for personalized therapeutic approaches.Keywords: Acute myeloid leukemia, Relapse, OMICS, Allogeneic hematopoietic stem cell transplant